Interferons (IFN) exert anti-viral and immunomodulatory effects through their induction of hundreds of interferon stimulated gene (ISGs) products. While several of these genes, such as PKR, RNaseL, and Mx have been studied, hundreds of these genes still have no known function. This is a proposal to define the physiologic importance and mechanisms of action of one of these unknown genes, IFN stimulated gene 15 (ISG15). ISG15 is an IFN induced protein that contains two ubiquitin like domains. In addition to coupling to proteins in IFN stimulated cells, ISG15 is released from IFN treated cells and found in the serum of IFN treated patients. Released ISG15 acts as a cytokine that triggers natural killer (NK) cell proliferation and enhanced killing, IFNg secretion from PBMC, and dendritic cell (DC) differentiation. We have found that ISG15 has antiviral effects in vivo, and that ISG15 expression in tissues (spleen and liver) is induced by virus infection. Importantly, we have found ISG15 in the serum during acute virus infection, consistent with a role as a cytokine. Together these data lead us to the hypothesis that ISG15 is a cytokine that acts during IFN responses to modulate the function of DCs and NK cells that are critical for innate and potentially adaptive immune responses. The goal of this proposal is to test this underlying hypothesis via three Aims. Studies in Aim 1 are designed to evaluate if ISG15 plays an important immunologic or anti-viral role in vivo. In particular, we will complete the generation of an ISG15 knockout mouse and determine if these mice have altered resistance to viral infection. The studies in Aim 2 are designed to determine if ISG15 functions as an extracellular cytokine to regulate immune responses. We will determine the biochemical nature of ISG15 isolated from sera of infected mice, determine the structural domains of ISG15 required for its anti-viral activity, and establish an in vitro assay for ISG15 cytokine function. The studies in Aim 3 are designed to identify the ISG15 receptor. The results obtained from these studies should provide further insight into the functional significance of ISG15, and provide a potential mechanism by which it exerts its antiviral activity. [unreadable] [unreadable] [unreadable]